Journal: bioRxiv

Article Title: RNA polymerase loss by nuclear rupture drives LMNA cardiomyopathy

doi: 10.64898/2026.04.03.716433

Figure Lengend Snippet: A) Immunofluorescence for bromouridine (BrU) after cardiomyocytes were cultured with BrU for 2 hr to label nascent RNAs. One binucleated cardiomyocyte per image, with nuclei zoomed in. Texts and icons below images indicate nuclear states. Scale bar: 5 μm. All data in , except , are from cardiomyocytes freshly isolated from mice at day 14 post tamoxifen. B) Nuclear BrU intensity by nuclear states after 1-hr BrU incubation. Density and box plots: BrU intensity distribution and interquartile range. Circles: mean intensity within biological replicates (color coded). Asterisks: P < 0.05 from t -tests on linear regression-estimated means with mouse-clustered standard errors. Underlying data: 244 intact nuclei from 4 WT mice, 275 intact, 111 ruptured, 104 resealed nuclei from 3 Lmna CKO mice. C) Relationship between nuclear BrU intensity and nuclear NLS–tdTomato intensity in all types of nuclei in Lmna CKO cardiomyocytes (519 nuclei from 3 mice). Line: simple linear regression fit with 95% confidence interval. R: Pearson correlation coefficient. P: t -test p -value on linear regression-estimated means with mouse-clustered standard errors. See for individual replicates. D) Immunofluorescence for RNA polymerase II (Pol II) in cardiomyocytes. Scale bar: 5 μm. E) Nuclear Pol II intensity by nuclear states. Underlying data: 285 intact nuclei from 3 WT mice, 180 intact, 133 ruptured, 136 resealed nuclei from 3 Lmna CKO mice. Graph annotations and statistics as in (B) . F) Relationship between Pol II intensity and NLS–tdTomato intensity in all types of nuclei in Lmna CKO cardiomyocytes (495 nuclei from 3 mice). Graph annotations and statistics as in (C) . G) Pol II ChIP-seq and input read coverage in WT and Lmna CKO cardiomyocytes (3 mice per genotype). ChIP-seq signals are normalized to internal spike-in controls. H) Average Pol II ChIP-seq signals across 21,177 protein-coding genes. X-axis: 100 equally-spaced bins in gene bodies, 5 bins for 1 kb-upstream regions, and 10 bins for 2 kb-downstream regions. I) Statistical comparison of gene-body Pol II signals in Lmna CKO versus WT cardiomyocytes for 11,942 Pol II-bound genes. Pol II-lost or gained genes are defined at limma p -value < 0.05. J) Ten most enriched Gene Ontology terms among the 1,759 Pol II-lost genes in Lmna CKO cardiomyocytes, with three representative genes for each term. P: Metascape p -value. K) Gene expression state of Pol II-lost, Pol II-gained, and all other genes in Lmna CKO (n=5) versus WT (n=7) hearts. P, DESeq2 p -value. RNA-seq data from En et al. 2024. L) Summary of . Nuclear rupture causes transcriptional deficiency due to RNA Pol II loss.

Article Snippet: We used AAVpro 293T cells (TaKaRa, 632273) for MyoAAV production and BJ-5ta cells (human hTERT-immortalized foreskin fibroblast; ATCC, CRL-4001) for ChIP-seq spike-in controls.

Techniques: Immunofluorescence, Cell Culture, Isolation, Incubation, ChIP-sequencing, Comparison, Gene Expression, RNA Sequencing

Journal: bioRxiv

Article Title: RNA polymerase loss by nuclear rupture drives LMNA cardiomyopathy

doi: 10.64898/2026.04.03.716433

Figure Lengend Snippet: A) Number of Pol II ChIP-seq and input sequencing reads aligned to the mouse genome (experimental) or the human genome (spike-in control). Scale factors are computed by spike-in control reads and sequencing depths and used to normalize Pol II ChIP-seq signals. All data in are derived from mice at 2 weeks post tamoxifen. B) Pol II ChIP-seq read coverage in all mouse genes stratified by gene-body Pol II coverage. Genes with Pol II coverage greater than or equal to 100 (2 in Log 10 ) were considered Pol II-bound (11,942 genes). C) Pol II ChIP-seq read coverage in all human genes, derived from the spike-in control chromatin. D) Gene-body Pol II ChIP-seq read coverage between every pair of biological replicates. E) Principal Component Analysis (PCA) of gene-body Pol II coverage in 11,942 Pol II-bound protein-coding genes. F) Cumulative fraction of 1,759 Pol II-lost genes and all other genes (y-axis) along the scale of differential gene expression between Lmna CKO hearts and wild-type hearts (x-axis). P, Kolmogorov-Smirnov test p -value comparing log 2 fold change of gene expression between Pol II-lost genes and all other genes. G) Gene expression state of Pol II-lost genes, Pol II-gained genes, and all other genes in the cardiomyocyte population in Lmna CKO (n=3) versus WT (n=3) hearts derived from single-nucleus RNA-seq in En et al. 2024. P, DESeq2 p -value. H) Same as F, but along the scale of differential gene expression between Lmna CKO and wild-type pseudo-bulk cardiomyocytes from the single-nucleus RNA-seq. P, Kolmogorov-Smirnov test p -value comparing log 2 fold change of gene expression between Pol II-lost genes and all other genes.

Article Snippet: We used AAVpro 293T cells (TaKaRa, 632273) for MyoAAV production and BJ-5ta cells (human hTERT-immortalized foreskin fibroblast; ATCC, CRL-4001) for ChIP-seq spike-in controls.

Techniques: ChIP-sequencing, Sequencing, Control, Derivative Assay, Gene Expression, RNA Sequencing

Journal: Nucleic Acids Research

Article Title: RPA hyperphosphorylation hinders the resolution of R-loops and G-quadruplex-associated R-loops during RAS-driven senescence

doi: 10.1093/nar/gkag331

Figure Lengend Snippet: Genome-wide analysis reveals loss of RNase H1 activity at RPA32-hyperphosphorylated R-loop–enriched regions in pre-RIS cells. ( A ) Schematic representation of the ChIP-seq and DRIP-seq experiments performed in the indicated cells at the indicated time after 4-OHT treatment. ( B ) Metaplots of RNase H1 ChIP-seq levels in the indicated cells, with respect to TSS and TES. ( C ) Histogram representing the percentage of genome coverage of γH2AX and RNase H1 enriched peaks in the indicated cells. ( D ) Volcano plot showing RNase H1 peaks that are significantly enriched or depleted in the comparison between the RPA70 + RAS and the H2B + RAS cells. ( E ) Histogram showing the percentage of γH2AX + genomic regions overlapping with areas of RNase H1 binding loss in H2B + RAS cells compared with the RPA70 + RAS condition, and vice versa. ( F ) Metaplot showing the log2 ratio of γH2AX signal in H2B + RAS with respect to RPA70 + RAS cells within 6kb around the RNase H1 peaks lost in H2B + RAS cells. ( G ) Histogram and metaplot showing the percentage of γH2AX + genomic regions in H2B + RAS cells overlapping with genomic regions exhibiting increased TrAEL-seq read density in IMR90 RAS (≥1.5-fold enrichment after 3 days of RAS expression relative to day 0), consistent with replication fork pausing or altered fork dynamics (GSE299123). ( H ) Histogram showing the percentage of TrAEL-seq–enriched regions in HRAS-expressing cells overlapping with genomic loci characterized by R-loop accumulation in BJ + RAS pre-RIS cells. ( I ) Histogram showing that RNase H1 peaks lost in H2B + RAS cells are associated with a depletion of RPA32 binding compared to RPA70 + RAS cells. ( J ) Metaplot of log2 ratio of RPA32 phosphorylation rate (pRPA32/RPA32) signal in H2B + RAS with respect to RPA70 + RAS cells within 6kb around the RNase H1 peaks lost in H2B + RAS cells. ( K ) Venn diagram showing that 25.3% of R-loops exclusive to the pre-RIS condition do not exhibit overlap (1 nucleotide) of RNase H1 binding in H2B + RAS cells, while 74.6% are characterized by RNase H1 association. ( L ) Venn diagram showing that 25.3% of R-loops exclusive to the pre-RIS condition that do not exhibit overlap (≥1 nucleotide) with RNase H1 binding in H2B + RAS cells show overlap with 38.3% of G4 regions. ( M ) Metaplot showing the hyperphosphorylation of RPA32 in H2B + RAS cells with respect to RPA70 + RAS within 6 kb around the center of the peaks of R-loops exclusive to the RIS condition and characterized by RNase H1 binding. For reference, a heatmap depicting the TrAEL-seq signal in IMR90 cells expressing RAS at the indicated time points is shown. ( N, O ) Distribution of R-loops, G4s, γH2AX and pRPA32 phosphorylation rate in RIS (BJ + RAS) and RIS-escape achieved by RPA70 overexpression (RPA70 + RAS). QPS are indicated. In C, E, G, H, I data are expressed as mean ± SD; two independent sequencing each coming from three biological replicates. * P < 0.05, ** P < 0.01. Pairwise t-test was applied to indicated comparisons.

Article Snippet: DNA SMART ChIP-Seq Kit (Takara) was used for library generation of samples subjected to Illumina sequencing.

Techniques: Genome Wide, Activity Assay, ChIP-sequencing, Comparison, Binding Assay, Expressing, Phospho-proteomics, Over Expression, Sequencing

Journal: Nucleic Acids Research

Article Title: RPA hyperphosphorylation hinders the resolution of R-loops and G-quadruplex-associated R-loops during RAS-driven senescence

doi: 10.1093/nar/gkag331

Figure Lengend Snippet: RAS-induced senescence is characterized by the accumulation of irreparable DNA damage, DNA:RNA hybrids and unsuccessful loading of BRCA1. A. Immunoblot analysis of the indicated proteins in BJ-hTERT/RAS-ER cells, expressing the indicated transgenes and treated or not as indicated with 4-OHT (1µM). % of SA-β-gal positivity is indicated. B. Quantification of SA-β-gal- positive cells and S-phase entering cells (BrdU assay, BrdU pulse of 3 h) in BJ-hTERT/RAS-ER Hygro cells or BJ-hTERT/RAS-ER HDAC4 cells, treated (+RAS) or not (-RAS) with 4-OHT for the indicated days (D2 = day2, D8 = day8) to induce HRAS G12V expression. Mean ± SD; n = 4. * P < 0.05, ** P < 0.01 and *** P < 0.001 (Dunn’s Multiple Comparison Test with respect to -RAS D2). Pairwise t-test was applied to indicated comparisons. C. Representative microscopic images of SA-β-gal stained BJ-hTERT/RAS-ER Hygro cells or BJ-hTERT/RAS-ER HDAC4 cells at the indicated time (days) after 4OHT treatment (Bar = 40 μm). D. Dot blot analysis using S9.6 antibody to detect R-loops and AE-2 antibody to detect dsDNA. Around 100 and 500 ng of nucleic acids extracted from the indicated cells grown for 2 days in presence or absence of 4-OHT were treated or not with 10u of RNase H and spotted on nitrocellulose film. E. Quantification of Dot blot shown in D. Mean ± SD; n = 3. F. Schematic representation of the ChIP-seq and DRIP-seq experiments performed in the indicated cells at the indicated time after 4-OHT treatment. G. Histogram representing the percentage of genome coverage of RNA PolII Ser5, γH2AX, BRCA1, R-loops and G4s enriched peaks in the indicated cells. Mean ± SD; two independent sequencing each coming from three biological replicates. * P < 0.05, ** P < 0.01 and *** P < 0.001. (Dunn’s Multiple Comparison Test with respect to -RAS D2). Pairwise t-test was applied to indicated comparisons. H. P -value and q-value of pathway enrichment analysis performed by using MSigDB (RRID:SCR_016863) on genes associated to DNA:RNA hybrids found exclusive of BJ + RAS condition.

Article Snippet: DNA SMART ChIP-Seq Kit (Takara) was used for library generation of samples subjected to Illumina sequencing.

Techniques: Western Blot, Expressing, BrdU Staining, Comparison, Staining, Dot Blot, ChIP-sequencing, Sequencing

Journal: Nucleic Acids Research

Article Title: RPA hyperphosphorylation hinders the resolution of R-loops and G-quadruplex-associated R-loops during RAS-driven senescence

doi: 10.1093/nar/gkag331

Figure Lengend Snippet: The co-occurrence of R-loops and G-quadruplex structures characterized the RIS condition. ( A ) Metaplot and heatmap of DRIP-seq signal 6kb around the 11 439 genomic regions with significant enrichment of DNA:RNA hybrids in BJ + RAS cells compared with BJ + RAS/HDAC4 cells. ( B ) Metaplot of RNA PolII Ser5, γH2AX, BRCA1 and G4 ChIP-seq signals within 6 kb around the 11 439 R-loops characterizing the pre-RIS condition. ( C ) Metaplot of log2 ratio of γH2AX signal in BJ + RAS with respect to BJ + RAS/HDAC4 cells within 6 kb around the 11 439 R-loops characterizing the RIS condition. ( D ) Histogram reporting the percentage of RIS-specific R-loops co-localizing with G4s within the indicated genomic interval. ( E ) Histogram reporting the genomic distribution of R-loops and “G-loop like” structures (defined as containing a G4 within 3 kb from DNA:RNA hybrids detected in DRIP-seq). ( F ) Histograms reporting the percentage of RIS-specific R-loops with G4 (defined as containing a G4 within 3kb from DNA:RNA hybrids detected in DRIP-seq) or without G4 co-localizing with G4s withint the indicated genomic interval in the indicated cellular conditions. ( G ) Distribution of R-loops, RNA PolII Ser5 (RNP2 Ser5), G4s, γH2AX, and BRCA1 on chromosome 12 and 16 in the indicated cells expressing or not RAS and HDAC4, as indicated. The highlighted region represents the genomic locus where a significant enrichment of the DRIP-seq signal is detected in BJ + RAS cells. At the PDXDC1 locus, enrichment encompasses multiple distinct regions within the highlighted interval, which spans approximately 15 kb. In B and F, data are expressed as mean ± SD; two independent sequencing each coming from 3 biological replicates. * P < 0.05, ** P < 0.01. Pairwise t-test was applied to indicated comparisons.

Article Snippet: DNA SMART ChIP-Seq Kit (Takara) was used for library generation of samples subjected to Illumina sequencing.

Techniques: ChIP-sequencing, Expressing, Sequencing

Journal: Nucleic Acids Research

Article Title: RPA hyperphosphorylation hinders the resolution of R-loops and G-quadruplex-associated R-loops during RAS-driven senescence

doi: 10.1093/nar/gkag331

Figure Lengend Snippet: G-loops exhibit resistance to RNase H1-mediated cleavage, suggesting a structural configuration that impairs enzymatic accessibility. ( A ) Motif discovery analysis reporting the three most frequent motifs found enriched in G4 ChIP-seq in BJ + RAS cells and co-localizing with R-loops within 3 kb. Each motif was analyzed using G4Hunter to identify the optimal sequence containing a putative QPS. The G4Hunter score is reported along with the predicted tetrads. ( B ) Plot reporting the distances in BJ + RAS of the G4s (corresponding to the three motifs found in Fig. ) with respect to the closest R-loop, divided according to their co-occurrence on either the template or non-template strand. ( C ) Heatmap showing the signal intensity of G4 structures and DRIP-seq in BJ + RAS cells, relative to the indicated genomic coordinates and the presence of QPSs marked by red arrows. In the case of the KRAS locus, these correspond to the well-characterized G4 regions referred to as ‘near’, ‘mid’, and ‘far’. ( D ) Metaplot of DRIP-seq and G4, RNA PolII Ser5 and BRCA1 ChIP-seq signals in BJ + RAS and BJ + RAS/HDAC4 cells within 6kb of “G-loop like” structures (R-loops co-occurring with G4 in non-template strand) identified in BJ + RAS cells. ( E ) The in vitro assay was performed by incubating equimolar amounts (800 pmol) of Cy5.5-labeled R-loops, G-loops containing the G4 motif “A” (ref. Fig. ) or DNA:RNA duplex (Cy5.5 labeling at 5′ of RNA) with increasing amounts of RNase H1. Native gel electrophoresis was performed to separate the various species of nucleic acids, which were schematized on the side. The fluorescence of the Cy5.5 labeled RNAs was acquired with the fluorescence reader and then the gel was stained with EtBr to detect the DNA duplexes with the transilluminator. ( F ) Histogram representing the ribonuclease efficiency of RNase H1 against synthetic R-loop and G-loop substrates (increasing amounts: 80, 160, and 400 pmol). Lanes corresponding to gel electrophoresis (in E) are indicated. Structures containing G4 in displaced DNA strand are in black (lanes 8–12). ( G ) Histogram representing the increase in fluorescence of Thioflavin T in the presence of substrates containing G4. Fluorescence emission was collected at 485 nm after excitation at 425 nm and expressed as increase of fluorescence quantum yield at 485 nm in the presence of G4-forming sequences in displaced DNA strand (lanes 8–12, increasing amounts: 80, 160, and 400 pmol). ( H ) Dot blot analysis using BG4 antibody to detect G4. Increasing amount of the indicated synthetic DNA:RNA structures (DNA:RNA heteroduplex not containing G4, R-loop not containing G4, G-loop with motif A, G-loop with 32R G4) were spotted on nitrocellulose film. RNA was Cy5.5 labeled at 5′. Cy5.5 fluorescence was used as loading control. ( I ) Fluorescence anisotropy binding assays showing the interaction between catalytically inactive RNase H1 (deadRNase H1) and the indicated nucleic acid substrates. Binding curves were fitted to determine the apparent dissociation constants ( K d ) for each substrate. In F and G, data are expressed as mean ± SD; n = 4. * P < 0.05, ** P < 0.01, *** P < 0.05. Pairwise t-test was applied to indicated comparisons.

Article Snippet: DNA SMART ChIP-Seq Kit (Takara) was used for library generation of samples subjected to Illumina sequencing.

Techniques: ChIP-sequencing, Sequencing, In Vitro, Labeling, Nucleic Acid Electrophoresis, Fluorescence, Staining, Dot Blot, Control, Binding Assay

Journal: bioRxiv

Article Title: Modulation of rob expression accelerates development of antibiotic resistance in Yersinia enterocolitica

doi: 10.64898/2026.02.23.707304

Figure Lengend Snippet: (A–D) ChIP-seq profiles showing Rob binding at the promoter regions of meoA (A), tolC (B), mlaF (C), and atpI (D). Blue tracks represent normalized ChIP-seq signal in the control strain (top) and Rob-tagged strain (bottom). Gene orientations are indicated by arrows; scale bar, 500 bp. (E) Schematic model of Rob-dependent transcriptional regulation. Rob binding upstream of target operons activates genes involved in outer membrane permeability ( ompF/ompD ), multidrug efflux ( tolC–acrAB2 ), and phospholipid transport ( mla operon). (F) Relative expression of selected Rob regulon genes measured by qPCR, shown as fold change normalized to wild type (WT). Data represent mean ± SD. The red dashed line indicates WT expression level. (G) Corresponding fold changes in gene expression derived from RNA-seq analysis, normalized to WT, confirming global upregulation of Rob target genes. (H) Efflux activity measured over time using a fluorescence-based assay. Rob mutant exhibits significantly higher efflux compared to the WT. Data are shown as mean ± SD; **** indicates P < 0.0001.

Article Snippet: Immunoprecipitated DNA was subjected to ChIP-seq library preparation using Novogene’s standard workflow, including end repair, A-tailing, Illumina adapter ligation, size selection, and PCR amplification, and sequenced by Novogene (Munich, Germany) on an Illumina NovaSeq X Plus platform with paired-end 150 bp reads.

Techniques: ChIP-sequencing, Binding Assay, Control, Membrane, Permeability, Expressing, Gene Expression, Derivative Assay, RNA Sequencing, Activity Assay, Fluorescence, Mutagenesis

Journal: Molecular cell

Article Title: Unbalanced chromatin binding of Polycomb complexes drives neurodevelopmental disorders

doi: 10.1016/j.molcel.2026.01.023

Figure Lengend Snippet: (A) RING1 and RNF2 variants (top). Reported variants in ClinVar and cancer-related somatic (COSMIC) mutations in RING1 and RNF2 genes (bottom). Metadome plots (middle) represent the level of predicted intolerance for amino acid change in RING1A and RING1B. For COSMIC, only positions of interest are shown as labels. Circle size represents the number of patients reported. (B) ColabFold predictions of RING1A and RING1B variants in altering interaction with PCGF proteins. (C) WBs of dKO-RING1A/B cells expressing HA-tagged WT and mutant RING1A and RING1B. Vinculin and histone H3 served as loading controls. n = 3 independent experimental replicates. (D) Possible mechanisms of deleterious variants that result in a decrease or absence of H2AK119ub. (E) Partial protein sequence alignments of a subset of RING1B homologs. The conserved RING1B-R70 residue corresponds to C. elegans R181 and is indicated by a star. Conserved zinc-coordinating residues, blue ; required for stabilizing the E2 enzyme-E3 ligase interaction in mammals, red ; required for binding to the nucleosome in mammals, green predicted to be important for the RING1B:PCGF4 interaction, magenta 47; and predicted to mediate β sheet interactions, cyan. * indicates identical residues, and : and. indicate residues with strongly and weakly similar physicochemical properties, respectively. The secondary structure of SPAT-3 and H. sapiens RING1B is shown below. (F) WBs of H2AK119ub in the indicated genotypes. The dilution factor is 1:3. The spat-3(mgw26) allele is a full deletion of the spat-3 coding region. Quantification of H2AK119ub and SPAT-3 isoform A is normalized to loading controls (histone H3/actin) and shown relative to the sample indicated by an asterisk. ND, not detectable. (G) WBs in dKO-RING1A/B cells stably expressing HA-RING1B WT or HA-RING1B R70H . Vinculin and histone H2A and H3 served as fractionation controls. n = 3 independent experimental replicates. (H) Normalized H3K27me3 Cut&Run signal (two independent experimental replicates) in cells treated with 1 μM of vehicle (DMSO) or GSK343 for 72 h. See also and .

Article Snippet: Ring1b (ChIP-seq) , Cell Signaling Technology , Cat# 5694, RRID:AB_10705604.

Techniques: Expressing, Mutagenesis, Sequencing, Residue, Binding Assay, Stable Transfection, Fractionation

Journal: Molecular cell

Article Title: Unbalanced chromatin binding of Polycomb complexes drives neurodevelopmental disorders

doi: 10.1016/j.molcel.2026.01.023

Figure Lengend Snippet: (A) Strategy to generate Rnf2 WT/R70H ESCs by homologous recombination. (B) DEG from WT and two clones of Rnf2 WT/R70H ESCs (log 2 fold > 2, q < 0.01). n = 2 independent experimental replicates. (C) GO of upregulated genes in Rnf2 WT/R70H ESCs. (D) Heatmaps of Ring1b, H3K27me3, and H2AK119ub ChIP-seq (average signal of two independent experimental replicates) in WT and clone #1 of Rnf2 WT/R70H ESCs. (E) Strategy to generate HA and FLAG-tagged Rnf2 alleles by CRISPR-Cas9 in WT and Rnf2 WT/R70H ESCs. (F) Normalized Ring1b WT and Ring1b R70H Cut&Run signals in WT and Rnf2 WT/R70H ESCs. Signal was generated from two biological replicates from two independent WT and Rnf2 WT/R70H clones. HA and FLAG Cut&Run signals were merged (average of 4 replicates) to avoid potential bias from the HA and FLAG antibodies’ efficiency. (G) Anti-FLAG IPs in Rnf2 HA-WT/FLAG-R70H and Rnf2 FLAG-WT/HA-R70H ESCs followed by LC-MS/MS in three independent experimental replicates. Results are normalized to IgG as a negative control. Volcano plot shows proteins enriched or weakened in FLAG-Ring1b R70H compared with FLAG-Ring1b WT from Rnf2 WT/R70H ESCs. (H) Heatmaps of Cbx7 and Pcgf2, Rybp, Mtf2/Pcl2, and Jarid2 ChIP-seq (average signal of two independent experimental replicates) in WT and clone #1 of Rnf2 WT/R70H ESCs. (I) Genome browser screenshots of ChIP-seq from (H). (J) Mutabind2 scores upon the human RING1B R70H variant vs. full length and lacking their IDR, PCGF1-6 using AlphaFold and ColabFold. (K) Full-length Pcgf2 or lacking the IDR used in (L). (L) Anti-HA IPs followed by WBs against HA, Phc1, and Ring1b in WT and Rnf2 WT/R70H ESCs expressing HA-Pcgf2 WT or HA-Pcgf2 ΔIDR . (M) Model of PRC1/2 recruitment in Rnf2 WT/R70H ESCs. See also .

Article Snippet: Ring1b (ChIP-seq) , Cell Signaling Technology , Cat# 5694, RRID:AB_10705604.

Techniques: Homologous Recombination, Clone Assay, ChIP-sequencing, CRISPR, Generated, Liquid Chromatography with Mass Spectroscopy, Negative Control, Variant Assay, Expressing

Journal: Molecular cell

Article Title: Unbalanced chromatin binding of Polycomb complexes drives neurodevelopmental disorders

doi: 10.1016/j.molcel.2026.01.023

Figure Lengend Snippet: (A) Protocol to generate and differentiate NPCs. (B) Heatmap of DEG from ESCs vs. NPCs vs. differentiated NPCs (log 2 fold > 4, q < 0.01) and between WT and two clones of Rnf2 WT/R70H ESCs, NPCs, and 12-day-old differentiated NPCs (log 2 fold > 2, q < 0.01). n = 2 independent experimental replicates. (C) IFs of neuronal markers in WT and Rnf2 WT/R70H differentiated NPCs. n = 3 independent experimental replicates. Scale bar, 10 μm. (D) WBs of PRC1 subunits from WT ESCs and Pcgf2 KO and Rnf2 WT/R70H with and without Pcgf2 . (E) Pictures of NPCs derived from cells in (D). n = 4 independent experimental replicates. Scale bar, 400 μm. (F) Strategy to generate NPCs expressing Ring1b WT , Ring1b R70H , or Ring1b I53A/D56K in PRC1 CKO cells. (G) WBs of HA and Ring1b in PRC1 CKO cells expressing Ring1b WT , Ring1b R70H , or Ring1b I53A/D56K in the presence and absence of OHT treatment for 48 h. Vinculin was used as a loading control. (H) Pictures of NPCs derived from cells in (G) in a constant presence of OHT treatment. n = 2. Scale bar, 400 μm. (I) WBs of Nanog, Pax6, and H2AK119ub in NPCs derived from PRC1 CKO cells expressing Ring1b WT , Ring1b R70H , or Ring1b I53A/D56K . Vinculin was used as a loading control. (J) UMAP plots of scRNA-seq from 16-day-old WT and two clones of Rnf2 WT/R70H differentiated NPCs. n = 2 independent experimental replicates. (K) Cell type proportions of cells from (E). * p < 0.05. ANOVA test. (L) KEGG pathway of WT and two clones of Rnf2 WT/R70H NPCs. See also .

Article Snippet: Ring1b (ChIP-seq) , Cell Signaling Technology , Cat# 5694, RRID:AB_10705604.

Techniques: Clone Assay, Derivative Assay, Expressing, Control

Journal: Molecular cell

Article Title: Unbalanced chromatin binding of Polycomb complexes drives neurodevelopmental disorders

doi: 10.1016/j.molcel.2026.01.023

Figure Lengend Snippet: (A) Normalized Ring1b WT and Ring1b R70H Cut&Run signals in either WT or Rnf2 WT/R70H NPCs in WT Ring1b peak regions. Two biological replicates from two independent clones. Wilcox test. *** p < 0.001. (B) Normalized H3K27me3 and H2AK119ub Cut&Run signals in either WT or Rnf2 WT/R70H NPCs over all genome. Signal was generated from two biological replicates from two independent clones. Wilcox test. *** p < 0.001. (C) Genome browser screenshots of HA, FLAG, H3K27me3, and H2AK119ub Cut&Run (average signal between replicates) in the cells shown on the left. (D) Anti-FLAG IPs in Rnf2 HA-WT/FLAG-R70H and Rnf2 FLAG-WT/HA-R70H NPCs followed by LC-MS/MS in three independent experimental replicates. Results are normalized to IgG as negative control. Volcano plot shows proteins enriched or weakened in FLAG-Ring1b R70H compared with FLAG-Ring1b WT from Rnf2 WT/R70H NPCs. (E) RNA-seq heatmap of PcG target genes in ESCs that are upregulated in WT NPCs but retained PRC1/2 and are repressed in Rnf2 WT/R70H NPCs. #1 and #2 are two different Rnf2 WT/R70H ESC clones. On the right, GO from each cluster. Deseq2; Wald test (FC > 4), q < 0.05. (F) Simplified genome browser screenshots of Ring1b WT , Ring1b R70H , H3K27me3, and H2AK119ub Cut&Run in WT and Rnf2 WT/R70H NPCs. Ring1b signal in WT NPCs and Ring1b WT and Ring1b R70H signals in Rnf2 WT/R70H NPCs are from merging average signals HA and FLAG Cut&Run two replicates from two clones. (G) Normalized signal of Ring1b WT and Ring1b R70H Cut&Run signals as in (F) around the transcription start site (TSS) of genes from (E). (H) Normalized signal of H3K27me3 and H2AK119ub Cut&Run signals (average from two replicates) as in (F) around the TSS of genes from (E). (I) Normalized ATAC-seq signal (average from two replicates) in WT and Rnf2 WT/R70H ESCs and NPCs around the TSS of genes from (E). See also .

Article Snippet: Ring1b (ChIP-seq) , Cell Signaling Technology , Cat# 5694, RRID:AB_10705604.

Techniques: Clone Assay, Generated, Liquid Chromatography with Mass Spectroscopy, Negative Control, RNA Sequencing

Journal: Molecular cell

Article Title: Unbalanced chromatin binding of Polycomb complexes drives neurodevelopmental disorders

doi: 10.1016/j.molcel.2026.01.023

Figure Lengend Snippet: (A) PCA from ATAC-seq from two independent biological replicates of WT and Rnf2 WT/R70H ESCs and NPCs. (B) Genome browser of ATAC-seq signal (average of two replicates) from WT and Rnf2 WT/R70H ESCs and NPCs. (C) RT-qPCR of pluripotency genes and NPC markers in WT and Rnf2 WT/R70H ESCs and NPCs. n = 3. #1 and #2 represent two clones of Rnf2 WT/R70H ESCs. *** p < 0.005, **** p < 0.001 by ANOVA test. (D) WB of Pax6 in WT and clone #1 of Rnf2 WT/R70H ESCs and NPCs. Vinculin served as a loading control. (E) ATAC-seq peaks reduced in Rnf2 WT/R70H NPCs and HOMER analysis. (F) Normalized expression of genes from (E) in WT and clones #1 and #2 of Rnf2 WT/R70H NPCs. *** p < 0.001. NS, not significant. Wilcox test. (G) ATAC-seq specific peaks in Rnf2 WT/R70H NPCs and HOMER analysis. (H) Normalized expression of genes from (G) in WT and clones #1 and #2 of Rnf2 WT/R70H NPCs. *** p < 0.001. NS, not significant. Wilcox test. (I) ATAC-seq signal in WT and Rnf2 WT/R70H NPCs at Sox2- or Sox3-occupied sites in WT NPCs. Sox2 and Sox3 ChIP from Bergsland et al. (J) Genome browser of ATAC-seq signal from WT and Rnf2 WT/R70H ESCs and NPCs as well as Ring1b WT and Ring1b R70H Cut&Run signal in WT and Rnf2 WT/R70H NPCs. (K) Normalized expression and GO of genes occupied by Ring1b WT and Ring1b R70H and compacted. *** p < 0.001. NS, not significant. Wilcox test. See also .

Article Snippet: Ring1b (ChIP-seq) , Cell Signaling Technology , Cat# 5694, RRID:AB_10705604.

Techniques: Quantitative RT-PCR, Clone Assay, Control, Expressing

Journal: Molecular cell

Article Title: Unbalanced chromatin binding of Polycomb complexes drives neurodevelopmental disorders

doi: 10.1016/j.molcel.2026.01.023

Figure Lengend Snippet: (A) Strategy to generate Rnf2 WT/R70H ESCs by homologous recombination. (B) DEG from WT and two clones of Rnf2 WT/R70H ESCs (log 2 fold > 2, q < 0.01). n = 2 independent experimental replicates. (C) GO of upregulated genes in Rnf2 WT/R70H ESCs. (D) Heatmaps of Ring1b, H3K27me3, and H2AK119ub ChIP-seq (average signal of two independent experimental replicates) in WT and clone #1 of Rnf2 WT/R70H ESCs. (E) Strategy to generate HA and FLAG-tagged Rnf2 alleles by CRISPR-Cas9 in WT and Rnf2 WT/R70H ESCs. (F) Normalized Ring1b WT and Ring1b R70H Cut&Run signals in WT and Rnf2 WT/R70H ESCs. Signal was generated from two biological replicates from two independent WT and Rnf2 WT/R70H clones. HA and FLAG Cut&Run signals were merged (average of 4 replicates) to avoid potential bias from the HA and FLAG antibodies’ efficiency. (G) Anti-FLAG IPs in Rnf2 HA-WT/FLAG-R70H and Rnf2 FLAG-WT/HA-R70H ESCs followed by LC-MS/MS in three independent experimental replicates. Results are normalized to IgG as a negative control. Volcano plot shows proteins enriched or weakened in FLAG-Ring1b R70H compared with FLAG-Ring1b WT from Rnf2 WT/R70H ESCs. (H) Heatmaps of Cbx7 and Pcgf2, Rybp, Mtf2/Pcl2, and Jarid2 ChIP-seq (average signal of two independent experimental replicates) in WT and clone #1 of Rnf2 WT/R70H ESCs. (I) Genome browser screenshots of ChIP-seq from (H). (J) Mutabind2 scores upon the human RING1B R70H variant vs. full length and lacking their IDR, PCGF1-6 using AlphaFold and ColabFold. (K) Full-length Pcgf2 or lacking the IDR used in (L). (L) Anti-HA IPs followed by WBs against HA, Phc1, and Ring1b in WT and Rnf2 WT/R70H ESCs expressing HA-Pcgf2 WT or HA-Pcgf2 ΔIDR . (M) Model of PRC1/2 recruitment in Rnf2 WT/R70H ESCs. See also .

Article Snippet: MTF2 Polyclonal antibody (ChIP-seq) , PROTEINTECH , Cat# 16208-1-AP, RRID:AB_2147370.

Techniques: Homologous Recombination, Clone Assay, ChIP-sequencing, CRISPR, Generated, Liquid Chromatography with Mass Spectroscopy, Negative Control, Variant Assay, Expressing